title: Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis.
abstract: The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis-infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, UK.
authors.forename: Keriann M
authors.initials: KM
authors.lastname: Backus
authors.forename: Helena I
authors.initials: HI
authors.lastname: Boshoff
authors.forename: Conor S
authors.initials: CS
authors.lastname: Barry
authors.forename: Omar
authors.initials: O
authors.lastname: Boutureira
authors.forename: Mitul K
authors.initials: MK
authors.lastname: Patel
authors.forename: François
authors.initials: F
authors.lastname: D'Hooge
authors.forename: Seung Seo
authors.initials: SS
authors.lastname: Lee
authors.forename: Laura E
authors.initials: LE
authors.lastname: Via
authors.forename: Kapil
authors.initials: K
authors.lastname: Tahlan
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Barry
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
mesh_terms: D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014199:Trehalose / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D014199:Trehalose